Objective:To construct suitable vectors for the secretory expression of hirudin in Pichia pastoris.Methods:The α-facor-hirudin gene was amplified from pPIC9-hirudin by PCR and sub-cloned into PA0815.The multi-copy recombinant plasmid pA0815-(α-Hirudin)n was constructed.The recombinant was transformed into P.pastoris strain GS115 for induction expression and then the activity of secreted products was identified.Results:A new multi-copy vector pA0815-(αHirudin)n was successfully constructed and was capable of secreting recombinant hirudin efficiently,which was confirmed respectively by PCR and SDS-PAGE.The products possessed the activity of thrombin inhibitor.Conclusion:This result offers efficient P.pastoris stains for mass production of biological active hirudin.

OBJECTIVETo explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells.

METHODSLuciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3'-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay.

RESULTSThe expressions of PKM2 mRNA and protein were upregulated (5.12 +/- 0.35) times and (4.05 +/- 0.20) times respectively in the PC3 cells as compared with those in the RWPE-1 cells (P < 0.05). Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3'-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated (0.16 +/- 0.04) times (P < 0.05), while the PKM2 mRNA level was not changed significantly (P > 0.05), as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were (66.20 +/- 5.10)% vs (82.10 +/- 6.35)% at 24 hours (P < 0.05) and (49.34 +/- 2.37)% vs (70.10 +/- 5.80)% at 48 hours (P < 0.05).

CONCLUSIONmiR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.

Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Thyroid Hormones ; genetics ; metabolism ; Transfection

ObjectiveTo evaluate the value of tissue mitral annular displacement (TMAD) in the assessment of left ventricular long axis systolic function and the relationship of obese degree with left ventricular long axis systolic function in patients with abdominal obesity.MethodsThirty-eight abdominal obesity cases and thirty-four healthy cases were investigated using echocardiography.The images of apical four-chamber view and apical two-chamber view were obtained,systolic mitral annular displacement (MADs),mid-point of mitral annular displacement(MAD-midpt),mid-point of mitral annular normalized displacement(MAND-midpt) and mid-point of mitral annular biplanar normalized displacement(MABNDmidpt) were measured by the technique of TMAD.Their characteristics between patients with abdominal obesity and healthy group were compared,and the relationship of waist-hip ratio(WHR) and related indexes of MAD were analyzed.ResultsIn abdominal obesity group,the MADs at the four site were significantly decreased compared with control group (P＜0.001,respectively),the MAD-midpt and MAND-midpt of apical four-chamber view and apical two-chamber view were significantly decreased compared with control group (P＜0.001,respectively).The WHR related with MABND-midpt independently by the analysis of partial correlation(r=-0.697,P=0.000).ConclusionsThe left ventricular long axis systolic function were damaged in patients with abdominal obesity.The technique of TMAD could quantitatively assess the left ventricular long axis systolic function in patients with abdominal obesity.

OBJECTIVETo determine the effects of Ginkgo biloba extract (EGB) on major periodontal pathogens in subgingival plaque.

METHODSSixty patients with moderate to severe periodontitis were selected and randomly assigned to 3 groups: EGB group, a positive (periocline) and a negative control groups. Subgingival plaque samples were collected before treatment and 1 week, 2 months and 4 months after treatment. The detection rates of 4 major periodontal pathogens-Treponema denticola (Td), Tannerella forsythus (Tf), Prevotella intermedia (Pi), and Porphyromonas gingivalis (Pg)-were detected by polymerase chain reaction (PCR). Clinical indicators were examined before treatment, 3 and 6 months after treatment.

RESULTSEGB significantly decreased the detection rate of all the 4 pathogens 1 week after treatment, and then gradually increased at 2 and 4 months. EGB's inhibition effect was better than or comparable to periocline, except for Pg in short-term. The difference of plaque index (PLI) and bleeding index (BI) was not statistically significant among the groups, while for probing depth (PD) and attachment loss (AL), the difference was statistically significant between the EGB group and negative control group at 3 and 6 months after treatment.

CONCLUSIONEGB significantly inhibited major periodontal pathogens and can be used as an adjuvant for periodontitis treatment.

Adjuvants, Pharmaceutic ; pharmacology ; therapeutic use ; Bacteria ; drug effects ; isolation & purification ; Dental Plaque ; drug therapy ; microbiology ; Follow-Up Studies ; Ginkgo biloba ; chemistry ; Humans ; Periodontium ; drug effects ; microbiology ; pathology ; Plant Extracts ; pharmacology ; therapeutic use ; Polymerase Chain Reaction ; Treatment Outcome

OBJECTIVETo explore the feasibility of application of adipose-derived cells (ADCs) in reconstruction of tissue engineered cartilage in vitro.

METHODSAdipose tissue were obtained from human liposuction aspirate (19 cases, 31.5 +/- 5.8 years old). ADCs were isolated by collagenase digestion from liposuction aspirates. 3rd passage cells were seeded into PLGA scaffolds. The copolymer constructs were cultured in conditioned or non-conditioned medium in vitro for 4 weeks. The constructs were evaluated though gross morphology, histology, and immunohistochemistry.

RESULTSThe cell-polymer constructs kept its original shape in the induced group, but lost its original shape in the non-induced group. The scaffold group were collapsed. Histologically, the induced groups showed dense cellularity and lacunae-containing cells embedded in a basophilic matrix, while non-induced groups showed connective tissue-like morphology. Collagen and proteoglycan deposition was revealed by Massons's trichome and Safranin' O staining, and minor collagen II expression in the matrix was detected by immunohistochemistry staining in the induced group. They were all negative in the non-induced groups.

CONCLUSIONSAlthough ADCs included many kinds of cells, it is feasible to use ADCs as seeds cells for reconstruction of tissue engineered cartilage.

Adipocytes ; cytology ; Adipose Tissue ; cytology ; Adult ; Cartilage ; cytology ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Female ; Humans ; Stromal Cells ; cytology ; Tissue Engineering ; methods ; Tissue Scaffolds

The industry of germ-free animals has been a hot spot in research along with the rapid development of studies on the relationship between microbiota and host diseases. Because it is pathogen?free, and the high degree of simi?larity in anatomy, physiology, pathogenesis to humans, germ?free pig is considered a clinical relevant model to be widely used in life science research. Based on the current state of research of germ?free pig cultivation at home and abroad and the experimental studies carried out in our laboratory as well, this article gives a simple discussion on germ?free technique of domestic pigs.